ABHD5 is an important lipase-activating protein, but how it functions to control lipolysis is poorly understood. Screening of the NIH compound library identified compounds that directly bind and activate ABHD5 independently of known extracellular signaling pathways. By using these probes in molecular imaging assays, we discovered endogenous ligands that bind ABDH5 and regulate its lipase-promoting activity. This proposal will investigate this novel locus of lipolysis control using integrated cellular and molecular approaches. A greater understanding of how these endogenous and synthetic compounds regulate ABHD5 could lead to novel therapeutics for treatment of obesity-related disorders.